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目的构建成纤维细胞生长因子受体(fibroblast growth factor receptor 2,FGFR2)基因野生型和E731K突变型的真核表达载体,并进行鉴定。方法利用基因定点突变试剂盒,定点突变FGFR2基因,获得其E731K突变的突变型基因,通过设计含有XbaⅠ、XhoⅠ限制性内切酶识别序列的引物分别扩增FGFR2基因野生型和E731K突变型的cDNA,克隆至质粒pcDNA3.1-EGFP上,构建重组表达质粒pcDNA3.1-FGFR2-EGFP和pcDNA3.1-FGFR2E731K-EGFP,利用X-tremeGENE HP DNA将质粒转染至HEK293细胞,采用实时荧光定量PCR法及Western blot法检测FGFR2表达水平。结果经定点突变已获得野生型的突变型FGFR2基因,碱基序列与设计序列完全一致,cDNA第2191位碱基G突变为A。重组表达质粒pcDNA3.1-FGFR2-EGFP和pcDNA3.1-FGFR2E731K-EGFP经双酶切及测序鉴定证明构建正确。质粒转染HEK293细胞后,荧光显微镜下均可见绿色荧光蛋白表达;质粒pcDNA3.1-FGFR2-EGFP和pcDNA3.1-FGFR2E731K-EGFP转染组中FGFR2 mNRA和蛋白表达水平均明显高于质粒pcDNA3.1-EGFP转染组和空白对照组(P<0.05)。结论成功构建了FGFR2基因野生型和E731K突变型的真核表达质粒,并在HEK293细胞中成功表达,为进一步研究FGFR2基因奠定了基础。
Objective To construct and identify the eukaryotic expression vector of wild-type and E731K mutant of fibroblast growth factor receptor 2 (FGFR2) gene. Methods The gene of FGFR2 was mutated by site-directed mutagenesis and the mutant gene of E731K was obtained. The wild-type and E731K mutant cDNAs of FGFR2 gene were amplified by designing primers containing Xba Ⅰ and Xho Ⅰ restriction enzyme recognition sequences. The recombinant plasmid pcDNA3.1-FGFR2-EGFP and pcDNA3.1-FGFR2E731K-EGFP were constructed and transfected into HEK293 cells using X-tremeGENE HP DNA. Real-time fluorescence quantitative PCR Method and Western blot were used to detect the expression of FGFR2. Results The wild-type mutant FGFR2 gene was obtained by site-directed mutagenesis. The nucleotide sequence of the wild-type FGFR2 gene was exactly the same as that of the designed sequence. Recombinant plasmid pcDNA3.1-FGFR2-EGFP and pcDNA3.1-FGFR2E731K-EGFP double digestion and sequencing proved that the construction is correct. The expression of green fluorescent protein was observed under fluorescence microscope after transfection of HEK293 cells. The expression of mNRA and protein of FGFR2 in pcDNA3.1-FGFR2-EGFP and pcDNA3.1-FGFR2E731K-EGFP groups were significantly higher than that of pcDNA3. 1-EGFP transfection group and blank control group (P <0.05). Conclusion The eukaryotic expression plasmids of wild type and E731K mutant of FGFR2 gene were successfully constructed and successfully expressed in HEK293 cells, which laid the foundation for further study of FGFR2 gene.